When the rate of availability of adenosine triphosphate (ATP.sub.A) for use in satisfying the overall metabolic reactions in a cell is depressed below the level that must be maintained just to satisfy those cellular energy needs for vital metabolic processes, the cell becomes incapable of mitotic division and ultimately dies. The rate of change in the ATP pool size existing in a cell at a particular time is the difference between the rate at which ATP is being produced, primarily by oxidative phosphorylation (O/P) along the Respiratory Chain (RC) in the mitochondria, and the rate at which ATP is being used up (hydrolyzed) to provide for all the energy requirements of the cell. This energy is principally required for all the myriad anabolic and catabolic reactions in the metabolism of the cell, including powering of the "sodium pumps" of the pericellular membrane--whose collective action keeps the intracellular Na.sup.+ -concentration relatively low despite the continuous leakage of Na.sup.+ through the membrane into the cell from the high Na.sup.+ -concentration extracellular fluid. The fundamental pathway involved in ATP production and usage (hydrolysis) in all normal body cells is depicted in FIG. 1.
The abbreviations and symbols used in FIG. 1 and elsewhere throughout this application are explained in the following table. Definitions of the primary therapeutical factors, the metabolic effectors Defined Nutritional Regimen (DNR), Fatty Acid Blocking Agent (FAB), Amino Acid Blocking Agent (AAB), ATP-Availability Depressor Agent (AAD) and Lactate Export Blocking Agent (LEB) of the present therapy system are given in the section entitled "Definitions of the Primary Metabolic Effectors," infra.
TABLE I ______________________________________ Abbreviations and Symbols ______________________________________ AA amino acids AAB amino acid blocking agent AAD ATP-availability depressor agent AcCoA acetyl coenzyme A ADP adenosine diphosphate ATP adenosine triphosphate [ATP] intracellular ATP concentration ##STR1## rate of production or degradation of ATP ATPase adenosine triphosphatase ##STR2## rate of availability of ATP for use in cellular metabolism ATP.sub.EMP ATP produced in the EMP ##STR3## rate of ATP production in the EMP ATP.sub.G ATP produced by glycolysis ##STR4## rate of ATP production by glycolysis ##STR5## ##STR6## ##STR7## rate of ATP produced by O/P in RC ##STR8## overall rate of production of ATP by cell ##STR9## rate of utilization of ATP by cell ##STR10## rate of wasting of ATP by AAD ##STR11## ##STR12## ##STR13## ##STR14## -c "with" (cum) Ca calcium CAC Citric Acid Cycle ##STR15## rate of operation of the CAC Cl.sup.- chlorine ion CO.sub.2 ##STR16## -CoA coenzyme A cm centimeter CPK creatine phosphokinase d day DFA DNR + FAB + AAB combination dl deciliter (100 ml) DNP 2,4-Dinitrophenol DNR defined nutritional regimen EMP Embden-Meyerhof Pathway FA fatty acids FAB fatty acid blocking agent g gram ##STR17## rate of operation of the Glycolytic (or EMP) Pathway ##STR18## ##STR19## H hydrogen (atomic) hr hour I Iodine i initial value of a quantity (subscript) I.U. international unit KCl potassium chloride Kg kilogram LAC lactic acid (lactate) ##STR20## rate of production of lactic acid in a cell ##STR21## rate of export of lactic acid from a cell LEB lactate export blocking agent lO.sub.2 /d liters of O.sub.2 consumed metabolically, per day (24 hours) max maximum min minute Mg magnesium mg milligram ml milliliter Mn manganese Na.sup.+ sodium ion NaCl sodium chloride NADH reduced nicotinamide adenine dinucleotide ##STR22## rate of supply of NADH to the RC O.sub.2 molecular oxygen O/P oxidative phosphorylation (in RC) P phosphorus PFK phosphofructokinase pH intracellular pH (acidity measure) pH.sub.L lethal level of intracellular pH RC Respiratory Chain RC rate of operation of the RC (amount of NADH oxidized per unit time) Se selenium T.sub.3 triiodothyronine T.sub.4 thyroxine TH thyroid hormone (T.sub.4 and/or T.sub.3) UA uncoupling agent of O/P Zn zinc .mu.g microgram .uparw. increase (in a rate) .dwnarw. decrease (in a rate) RDA recommended daily allowance .sup.-s "without" (sine) ______________________________________
In normal (i.e., non-malignant) body ce nutritional component is glucose, from which the primary energy supply for synthesizing ATP is derived. Glucose is transformed by the sequential reactions of the Glycolytic or Embden-Meyerhof Pathway (EMP) into pyruvate. Only about 6% of the total energy available in the original glucose molecule is released in the form of ATP during degradation in the EMP. Subsequently, pyruvate is decarboxylated and forms acetyl coenzyme A (AcCoA) which then enters the Citric Acid Cycle (CAC) in the mitochondria. Here each acetate moiety, after first being incorporated into a molecule of citric acid, is broken down into CO.sub.2 and H with H appearing, inter alia, in molecules of reduced nicotinamide adenine dinucleotide (NADH) which then contain a large fraction of the energy contained in the original glucose. This NADH subsequently is oxidized in the mitochondrial Respiratory Chain with the ultimate production of H.sub.2 O by terminal reaction of the H with O.sub.2. This O.sub.2 is supplied by the normal vasculature. The energy obtained by the transport of electrons down the potential gradient of the RC, by a series of redox reactions, is used to produce the ATP of the cell. About 94% of the total energy available in the original glucose molecule is released in the form of ATP during degradation of the AcCoA in the CAC and oxidation of the associated NADH in the RC. Thus, in normal cells, the ATP-stored energy is obtained in the major proportion from nutritional glucose or from carbohydrates (i.e., starches and sugars) which yield glucose upon digestion. Some ATP-energy is obtained in normal cells from the oxidation, in the CAC and RC, of fatty acids and amino acids obtained from nutritional fats and proteins. When adequate glucose is available in the nutriment intake, however, the major ATP-energy needs of practically all normal cells are readily obtainable from glucose alone. The ATP produced in the EMP and RC enters the cellular "ATP Pool", from which it is continuously withdrawn at the net availability rate ATP.sub.A to supply the energy needs of total cellular metabolism including energy to power the membrane sodium pumps which keep the intracellular Na.sup.+ -concentration adequately low by the out-pumping of Na.sup.+.
This same general pattern of ATP generation and usage exists in malignant cells, but with one crucial difference (see FIG. 2). It has been extensively demonstrated that the malignant cells of practically all forms of malignant neoplasms possess a common, distinctive metabolic aberrancy, apparently manifested as an innate consequence of their transformation to the malignant state [Niemtzow, R. C. (Ed.), Transmembrane Potentials and Characteristics of Immune and Tumor Cells Chapter 9, CRC Press, Boca Raton, Fla., (1985)]. Under in vivo conditions, the malignant cells of essentially all forms of malignant neoplasms do not substantially convert pyruvate to AcCoA (see FIG. 2). The pyruvate instead is essentially quantitatively converted to lactate which is exported from the cell by an effective lactate transport system [Warburg, O., Uber den Stoffwechsel der Tumoren, Springer-Verlag, Berlin and New York (1926); Warburg, O., The Metabolism of Tumors Constable, London (1930); Burk, D., Cold Spring Harbor Symposia Quant. Biol. 7, 420 (1939); Busch, H., An Introduction to the Biochemistry of the Cancer Cell, Chapter 10, Academic Press, New York ( 1962); Racker et al., Science 209, 203 (1981); Spencer, T. L. et al., Biochem. J. 154, 405 (1976); Belt, J. A. et al., Biochem. 18 3506 (1979): Weinhouse, S., Cancer Res. 3, 269 (1955); Busch, H. et al., Cancer Res. 20 50 (1960); Busch, H., Cancer Res. 13 789 (1955); Busch, H. et al., J. Biol. Chem. 196, 717 (1952); Nyham, W. L. et al., Cancer Res. 16, 227 (-957); Cori, C. F. et al., J. Biol. Chem. 64, 11 (1925); Cori, C. G. et al., J. Biol. Chem. 65, 397 (1925); Warburg, O. et al., Klin. Wochschr. 5, 829 (1926); Muramatsu, M., Gann. 52, 135 (1961); Busch, H. et al., Cancer Res. 16, 175 (1956)]. The net consequence is that only a small fraction ( .about.6%) of the chemical energy in the glucose molecule can be extracted and used by the cancer cell, compared to that available to the normal cell, where glucose is totally oxidized [White, A. et al., Principles of Biochemistry, 5th Ed., p. 441 (1973)]. Since nutritional glucose is by far the most prominent and important source of normal cellular ATP energy under normal conditions, this transformation aberrancy puts the malignant cells at a great disadvantage regarding the maximal rates at which they can generate ATP from glucose oxidation via the CAC and RC. This metabolic defect is potentially particularly restrictive for the malignant cells, which generally need an especially abundant availability rate of ATP to support the active anabolic metabolism associated with the frequent mitosis characteristic of these proliferative cells.
However, malignant cells in vivo quite effectively circumvent this energy deficiency under usual nutritional conditions by readily oxidizing fatty acids and amino acids in the CAC and RC [Busch, H. (1962) supra: Medes, G. et al., Cancer Res. 17 127 (1957); Allen, A. et al., J. Biol. Chem. 212, 921 (1955); Emmelot, C. et al., Experientia 11, 353 (1955); Weinhouse, S. et al., Cancer Res. 13, 367 (1953); Weinhouse, S. et al., Cancer Res. 11, 845 (1951); Kitada, S. et al., Lipids 15 168 (1980); Spector, A. A., J. Biol. Chem. 240, 1032 (1965)]. Mitochondria possess a very efficient enzyme system capable of effecting the ".beta.-oxidization" of fatty acids directly to AcCoA, which then enters the Citric Acid Cycle and is oxidized exactly as AcCoA produced from oxidation of glucose in normal cells. The amino acids are, after initial deamination, similarly reduced to AcCoA or other intermediates of the CAC and then oxidized. Thus, some amino acids, after deamination and suitable transformation, which is readily accomplished by the enzyme systems of malignant cells, are capable of entering the Citric Acid Cycle directly at various intermediate points of the cycle [Busch, H. (1962), supra]. Consequently, although substantially deprived of the utilization of glucose as a primary energy source, the malignant cells make full use of the supply of the energy-rich fatty acids, and amino acids, all present in the plasma under usual nutritional intake levels.
Under conditions where the rate of production of ATP by oxidative catabolism of free fatty acids (FA) and amino acids (AA) via the CAC-RC is inhibited in cancer cells (e.g., because of a limited rate of substrate and/or oxygen supply, or presence of an O/P uncoupling agent), or the oxidatively derived ATP-availability rate is otherwise depressed (e.g., by inappropriately stimulated ATPase activity), the cells are able to compensate in part for this energy rate loss by strongly increasing the rate of glycolysis (GLY) per se. This increased GLY results in a pronounced rise in the rate of production of lactic acid (LAC.sub.P). The lactate must concomitantly be rapidly exported from the cell in order to prevent the intracellular pH from decreasing to a lethal level because of a buildup in the lactate concentration. Under usual physiological conditions, the lactate export rate (LAC.sub.E) capacity of cancer cells is much more than adequate to prevent such an intracellular lactate buildup [e.g., Spencer, T. L. et al. (1976) supra: Belt, J. A. et al. (1979) supra]. Consequently, the cancer cells can operate at relatively high GLY levels when energy is relatively unavailable from oxidative pathways of the CAC and RC.
In accordance with the present invention, the net availability rate of ATP, ATP.sub.A, for satisfying the overall metabolic requirements of malignant cells in the body is depressed to a level which is inadequate for the maintenance of the essential metabolic processes required for the continued viability of the cells, without substantially altering the normal ATP.sub.A level in normal cells of the body (see FIG. 3). The malignant cells are thus selectively subjected to a lethal energy deprivation, resulting in cellular death as a consequence of energy starvation. In addition, the present invention provides simultaneously and synergistically for the stimulation of the GLY in malignant cells to a maximum level while concomitantly effectively limiting the maximum LAC.sub.E capability of the cells by inhibition of the lactate export system. The malignant cells are thus selectively subjected to a second alternate lethal action in which cellular death occurs as a consequence of acidity buildup and the depression of the intracellular pH below the level permissible for continued viability.
The most preferred embodiment of the present invention consists of the concurrent administration of five primary metabolic effectors (AAD, LEB, DNR, FAB and AAB), with sites of action as depicted in FIG. 3. For purposes of present dicussion, these metabolic effectors are arbitrarily grouped into three regimens which are, for clarity of presentation, discussed in the order in which they individually act in the metabolic energy pathway of the cancer cells (FIG. 3). As is detailed subsequently, other regimens and combinations of these metabolic effectors, although not constituting the most preferred embodiment for clinical application, are still fully capable of effecting very significant oncolysis.
The first regimen of metabolic effectors (DNR, FAB, AAB) is designed to substantially limit the maximum rate at which NADH can be supplied (NADH) to the RC of the cancer cells in the body, thus substantially limiting the maximum rate at which ATP can be made oxidatively (i.e., by the CAC-RC) by the cells, without limiting the rate of NADH supply (NADH) in the normal cells of the body to any significant degree. The second regimen's metabolic effector (AAD) is designed to degrade a substantial portion of such ATP as is produced or is potentially producible by the cancer cells, thus making it unavailable for cellular metabolic requirements. The pronounced deficit in the overall ATP.sub.A imposed by the first and second parts of the therapy, relative to that necessary to supply just the minimal ATP rate requirements of the essential metabolic processes, ultimately reduces the ATP pool selectively in the cancer cells to a lethal level. The third regimen's metabolic effector (LEB) is designed to greatly inhibit the rate at which glycolytically produced lactate can be exported from the cancer cells. The strong ATP.sub.A deficiency imposed by regimen two (supra) causes a pronounced increase in the cellular GLY and consequent LAC.sub.p, thus synergistically insuring, in combination with regimen three of the therapy system, an ultimately lethal lactate buildup, which acts by producing a lethal depression of the intracellular pH. The concurrent use of the combination of the three regimens of the present therapy system thus provides two separate, but synergistically related, modes of achieving the destruction of cancer cells in the body, either of which may be the ultimate cause of lethality in a given cancer cell under different physiological conditions.
The first regimen (see FIG. 3) comprises the administration of a defined nutritional regimen (DNR) which consists essentially of a dietary regimen designed to maximize the use of nutritional glucose-yielding carbohydrates as a source of ATP energy, and to minimize the availability of nutritional fatty acids and amino acids for use as a source of ATP energy (FIG. 3). It also comprises the concurrent use of one or more fatty acid blocking agents or "fatty acid blockers" (FAB) and amino acid blocking agents or "amino acid blockers" (AAB) to inhibit the availability of oxidatively obtained (i.e., CAC-RC) ATP-energy from endogenously derived (body depot or plasma) free fatty acids and amino acids for use by the cancer cells.
The second regimen (FIG. 3) comprises the concurrent administration of one or more ATP-availability depressor agents or "ATP-availability depressors" (AAD) which, at adequate levels, results in the lowering or depression to a lethal level in the cancer cells of the net rate of the ATP, ATP.sub.A, actually available for satisfying cellular metabolic needs, by directly inhibiting the synthesis rate of ATP per se (e.g., by use of uncoupling agents of O/P) and/or inactivating or hydrolyzing ATP already synthesized (e.g., by use of ATPase-hydrolysis-activity enhancing agents). Administration of the AAD makes unavailable to the cancer cells a large fraction of the maximum potential cellular ATP production per unit time otherwise available, a maximum already severely limited by the reduced availability of NADH resulting from the restriction of energy availability from fatty acids and amino acids by the DNR, FAB and AAB of the first part, and results in cell death by energy starvation. Since the normal cells of the body can make full use of the abundant carbohydrate (glucose) supplied by the DNR for energy purposes, the only effect on the normal cells is an increase in O.sub.2 consumption rate (i.e., in increased RC); the potential ATP loss in the normal cells due to the AAD is fully compensated by a higher rate of glucose-derived NADH oxidation (NADH) by the respiratory chain, while the rate of actual ATP production and availability ATP.sub.A remains unchanged at its normal level.
The third regimen (FIG. 3) comprises the administration of one or more lactate export blocking agents or "lactate export blockers" (LEB) which results in a substantial reduction of the maximum rate at which lactate can be exported from the glycolyzing cancer cells in the body. The LEB blocks a substantial portion of the normal maximal lactate export rate capacity of the cancer cells and allows the lactate to build up in the cells adequately to produce a lethal pH level.
Applicant has previously disclosed a related method of effecting oncolysis comprising the use of a defined nutritional regimen (DNR) in combination with one or more O/P uncoupling agents (UA) [U.S. Pat. No. 4,724,234]. That therapy system may be considered as a special, restricted case of the present invention consisting of use of only a DNR and an AAD, wherein the AAD is specifically an uncoupling agent of cellular oxidative phosphorylation. Applicant has also previously disclosed [U.S. Pat. No. 4,724,230] a method for effecting oncolysis consisting of a combination of a DNR and one or more UA, and the concomitant use of fatty acid oxidation inhibiting agents ("FAOI" therein) which result in the inhibition of oxidation in cellular mitochondria of free fatty acids. That system may likewise be considered as a special, restricted case of the present invention, consisting of a DNR, FAB, and AAD, wherein the FAB is specifically an inhibitor of mitochondrial free fatty acid oxidation (FAOI) and the AAD is specifically an oxidative phosphorylation uncoupling agent (UA).
The potentiality of destroying cancer cells in vitro by depressing their intracellular pH to a lethal level by use of substances which inhibit lactate export has been previously addressed, based on in vitro experiments with cancer cell cultures [Johnson, J. H. et al. Biochemistry 19 3836 (1980)]. However, no clinical method of effecting oncolysis utilizing lactate inhibiting agents has heretofore been advanced. Ostensibly, this is because of the in vitro finding that cancer cells have an enormous reserve capacity for lactate export, relative to the usual rate of GLY (LAC.sub.P) at which they operate. Consequently, the lactate export capability must be almost totally blocked before any lactate buildup and pH decrease occurs [Spencer, T. L. et al. (1976), supra: Belt, J. A. et al. (1979), supra1. Such a high level of blockage would be most difficult to achieve and maintain in vivo. Moreover, it is known that the GLY level decreases significantly as the intracellular pH decreases [Wilhelm, G. et al. FEBS Lett. 17, 158 (1971), Belt, J. A. et al. (1979) supra; Suolinna, E.-M. et al., Cancer Res. 35, 1865 (1975)], thus making the required degree of blockage essentially total. Without such 100% blockage, the LAC.sub.p and hence the pH decline becomes self-limiting, and it is not possible generally to effect cancer cell death, even in vitro, by use of lactate export inhibiting agents alone. The present invention effectively overcomes these basic problems, since the pronounced depression of the cancer cell ATP.sub.A effected by the combination of parts one (DNR-FAB-AAB) and two (AAD) of the present invention raises the GLY and LAC.sub.P and maintains them at levels several fold greater than that normally existing (i.e., without such therapeutically imposed GLY stimulation) in cancer cells. Consequently, the high LAC.sub.p thereby effected not only ensures the maintenance of a high LAC.sub.p against the depressing tendency of a decreasing intracellular pH, but also thereby reduces substantially the degree of lactate export inhibition which must be effected in order to permit cellular lactate buildup and the intracellular pH to decrease to a lethal level. The present invention thus makes the use of lactate export blocking agents clinically practical and most efficacious.
Applicant has found in evaluative clinical treatment regimens administered according to the present invention utilizing far advanced human cancer patients having histologically verified malignancies representing a wide range of malignancy types (tongue, throat, stomach, cecum, colon, rectum, breast, ovary, uterus, lung, kidney, prostate, pancreas, lymphoma, melanoma, skin, marrow (leukemia), and bone) that very significant oncolysis is effected. These efficacious results were obtained with patients whose disease was found to be uncontrollable with conventional mitoxin chemotherapy and radiotherapy modalities. Throughout the treatment period of the individual patients, the clinical regimen was generally found to be free of discernable toxic side effects, and allowed a very high quality of life, despite the poor entry condition of most of the patients.
The therapy system of the present invention substantially avoids several of the traditional problems and limitations of conventional mitoxin chemotherapy. Mitoxin chemotherapy characteristically acts by the indiscriminate destruction of all mitotically active cells in the body, both normal and malignant. Because of this mass indiscriminate destruction of normal proliferative cells by mitoxin chemotherapy, a host of toxic and treatment-limiting side-effects are experienced, including anemia (marrow destruction), pronounced loss of cellular and humoral immune competence, decrease of blood platelets, gastrointestinal ulceration and denudation with bleeding, vomiting and diarrhea, destruction of salivary gland function, electrolyte imbalance, anorexia, loss of hair, abnormalities of the nervous system, kidney damage, skin rash, liver damage, abnormal heart beat, myocardial toxicity, and damage to the lungs. The present method of metabolic chemotherapy, because it does not adversely affect normal dividing cells in the body, is strikingly free of such toxic effects and therefore permits continued administration until potentially all malignant cells are destroyed, while simultaneously permitting a very high quality of life.
Similarly, since the present method does not destroy blastogenic lymphocytes of the immune system as does mitoxin chemotherapy, the body's immune competence remains unaltered, thus avoiding the pronounced decrease in resistance to infectious diseases usually seen in human patients undergoing mitoxin chemotherapy while maximally enhancing potential immunological cell-mediated and humoral attack on residual tumor cells.